The structure, assembly and permeability of tight junctions found between the plasma membranes of cultured canine kidney cells (MDCK) will be studied in order to define more precisely the factors controlling transcellular permeability of endothelia and epithelia. After trypsinization and replating on permeable collagen-coated nylon discs, the MDCK cells attach and form a polarized epithelium within 24 hours. The development of transepithelial electrical resistance, a measure of permeability, will be recorded during the 24 hour period or after disassembling the junctions with EGTA and allowing them to reassemble over a 4-6 hour period and compared to the ultrastructure of the developing tight junctions as determined by freeze-fracture electron microscopy in order to quantitate what structural properties contribute to transcellular permeability. Competent tight junctions will be disassembled with EGTA and then allowed to reassemble in the presence of inhibitors of microfilament and microtubule function in order to investigate the role of cytoskeletal structures in junctional reassembly. Finally, the role of the tight junctions in providing an intramembraneous barrier to the mobility of plasma membrane components will be studied by observing the redistribution of membrane proteins and lectin receptors utilizing immunocytochemistry both after disassembling the junctions and during the regeneration of a functional polarized epithelium.